Human induced pluripotent stem cells (hiPSCs) are currently widely used to model human biology and disease. Beyond this, they hold enormous potential for patient derived regenerative medicine. Unfortunately, female hiPSCs display defective X chromosome inactivation, thereby confounding experiments and rendering them unsuitable for therapeutic applications.
X chromosome inactivation (XCI) is the dosage compensation mechanism to equal X-linked gene dosage between XX females and XY males, where one of the female X chromosomes becomes almost completely transcriptionally silenced. XCI occurs in every cell of an adult female mammal, including humans, and is necessary for life. XCI is established by a long non-coding RNA called XIST, which is expressed from the inactive X chromosome and establishes the silent state by coordinating a repressive chromatin environment. Unfortunately, over a very short time in culture hiPSCs lose XIST expression, which is followed by a loss of repressive chromatin and reactivation of silent X-linked genes. This occurs in all hiPSC lines tested to date and means that each inactive X chromosome is in a varying state of decay, with an unknown repertoire of X-linked gene expression. This defective pattern of XCI is then maintained through differentiation.
Recently, a new method of creating hiPSCs was developed (Buckberry et al., Nature 2023). Known as Transient-Naïve-Treatment (TNT) reprogramming, this method creates hiPSCs with an epigenome better suited to pluripotency. We have found that TNT reprogrammed hiPSCs also have correct XCI, with maintained XIST expression and localisation to the X chromosome, the expected repressive chromatin environment and gene silencing. The correct XCI state is maintained in culture. This discovery identifies TNT reprogrammed hiPSCs as a more accurate and useful model of female human biology.